Cytokines are active in cell proliferation, differentiation and movement. At picomolar to nanomolar concentrations, they effect such activities as leukocyte migration and function, hematopoietic cell numbers, temperature regulation, acute response to infections, tissue remodeling and cell survival. Since cytokines are produced in groups and in patterns characteristic of a particular stimulus or disease, studies using antibodies or other drugs to modify the activity of a particular cytokine are beginning to elucidate the roles of individual cytokines in pathology and physiology.
Endothelial-monocyte activating polypeptide II (EMAP-II) was first identified by Kao J et al (1994 J Biol Chem 269:25106-25119) in the supernatant of murine methylcholanthrene A-induced fibrosarcomas (METH A) where it functions to activate host cells. The full length cDNA which Kao et al cloned has 1086 bp and three ATG codons. Using Kozak's rules, Kao et al selected the second ATG, at position 64, as the most probable start codon and predicted that translation would produce a polypeptide of 310 amino acids and 34 kDa.
The mature EMAP-II is a unique, leaderless, single chain polypeptide with a predicted molecular mass of 20 kDa which most likely results from processing of the larger polypeptide. Using a fragment of the murine cDNA as a probe, Kao et al identified a human cDNA from a monocyte library with 86% identity. Both the designated start codon and the upstream ATG are conserved in the human cDNA.
The processing of the mature murine and human EMAP-II proteins appears to be conserved and shared among several other processed cytokines including interleukin-8 (IL-8), IL-1.beta., and in von Willebrand antigen II. IL-1.beta., which is approximately the same size as EMAP-II, also lacks a classic signal peptide and must be cleaved by the cysteine protease IL-1.beta.-converting enzyme at a conserved aspartate residue in the P-1 position to yield the secreted, biologically active 17-kDa form. Both murine and human forms of EMAP-II display an aspartate in the P-1 position. Processing of EMAP-II in this manner would produce a 18 kDa polypeptide with 165 amino acids.
Kao et al (supra) reported that EMAP-II promotes thrombohemorrhage and increases the vascular permeability of tumors. Purified, recombinant mature EMAP-II activates endothelial cells (ECs) and results in elevation of cytosolic free calcium concentration, release of von Willebrand factor, induction of tissue factor, and expression of the adhesion molecules, E-selectin and P-selectin. Neutrophils (PMNs) exposed to EMAP-II also demonstrate elevated cytosolic free calcium concentration as well as myeloperoxidase generation and chemotactic mobility. EMAP-II also activates mononuclear phagocytes (MPs) by inducing expression of tumor necrosis factor-.alpha. (TNF), increasing MP tissue factor activity, stimulating chemotaxis, moderately inducing elaboration of IL-8 and slightly elevating cytosolic free calcium concentration. In addition, preliminary evidence indicates that MPs produce EMAP-II when treated with LPS which suggests that EMAP-II plays a role in endotoxin-mediated host responses and the proinflammatory cytokine cascade.
Mice given a systemic infusion of 10 .mu.g EMAP-II derived from Meth A tumors developed the following: 1) systemic toxicity, including lethargy, decreased food/water intake, and ruffled body hair; 2) pulmonary congestion, including labored, rapid respiration and arrest of circulating leukocytes and other inflammatory cells in the pulmonary vasculature with concurrent increase of myeloperoxidase activity; and 3) the appearance of TNF.alpha., interleukin-1 and- 6 in the plasma. In another study, Kao et al found that a single intra-tumor injection of EMAP-II into Meth A sarcomas induced acute thrombohemorrhage in 67% of the animals, infiltration of tumor with PMNs, and partial tumor regression. Although local injection of EMAP-II alone into a TNF-resistant murine mammary carcinoma had no effect, when injection of EMAP-II was followed 15 h later by systemic administration of TNF, the tumor regressed following acute inflammatory infiltration by PMNs and thrombohemorrhage. In pilot studies, Kao et al found that a similar priming effect occurred when the tumor bed of B16 melanomas and human fibrosarcomas was treated with EMAP-II.
Kao et al (1994 J Biol Chem 269: 9774-82) further suggest that the amino terminus of cytokines may have a role in chemoattractant activity and the mobilization of intracellular calcium. For instance, mutagenesis of amino acids in the NH.sub.2 -terminus of IL-8 rendered the molecule incapable of mobilizing calcium in neutrophils and lowered its ability to compete with native IL-8 for neutrophil IL-8 receptors. Kao J et al created a synthetic oligopeptide comprising 15 residues from the N-terminal region of EMAP-II. This peptide induced directional migration and elevation of cytosolic free calcium concentration in MPs and PMNs and stimulated peroxidase release in PMNs. In addition, oligopeptide-albumin conjugates injected into the mouse footpad elicited infiltration of inflammatory cells. In contrast, a peptide from the C-terminus of EMAP-II had no effect.
In binding assays, the NH.sub.2 -terminal oligopeptide bound specifically to MPs. Binding of I.sub.125 -labeled oligopeptide was saturable and blockable using either unlabeled EMAP-II derived peptides or intact EMAP-II. However, other peptides such as IL-1, TNF, and formyl-methionyl-leucinyl-phenylalanine had no blocking effect. When cross-linked to human MPs, I.sub.125 -EMAP-II derived peptide demonstrated a band, approximately 73 kDa, on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis supporting the existence of a specific receptor. In addition, specific binding of the derived peptide to human PMNs and murine RAW cells was demonstrated.
Cytokines could satisfy a need in the art by providing new ways to treat cancers. The systemic infusion of cytokines appears to destroy specific cell types in the vasculature of B16 melanomas and fibrosarcomas. This destruction results in diminished blood flow, increased permeability to host effector cells and increased thrombohemorrhage. Utilization of the response to cytokine infusion is of particular therapeutic interest and has great value in the treatment of those cancers which are resistant to conventional treatments.